This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Aldolase are ubiquitous enzymes found in all organisms, from prokaryotes to mammals. They have the ability to cleave carbon-carbon bonds. Their role is best known in glycolysis where fructose-1,6-bisphosphate (FBP) aldolase cleaves FBP to dihydroxyacetone-phosphate (DHAP) and glyceraldehy-3-phosphate. The catalytic role assigment to residues has beeen hampered by the lack of genuine reaction intermediate crystallographic structures. The aim of this project is to study the catalytic mechanism of the rabbit muscle aldolase, which is a class I fructose-1,6-bisphosphate aldolase. We are planning to use subtrate soaking experiments with native and mutant aldolase crystals to get insight about residues of the active site implicated in the catalysis. By using a combination of diverse soaking time, we want to trap catalytic reaction intermdiates among the reaction scheme, which is known to implicate the formation of 3 covalent intermediates. Time-resolved crystallography applied to rabbit muscle aldolase crystal soaked with ligands or substrates have been successfully used in the past years. Further insight into the catalytic mechanism of aldolase still possible by using this technique.